Current state of populations of Rhodiola rosea L. (Crassulaceae) in East Kazakhstan

Based on world experience, first, a modern assessment of the flora is needed to develop strategies for the conservation of ecosystems of rare and endangered plant species. A regional and global biodiversity strategy should focus on assessing the current state of bioresources. To preserve the biodiversity of the species and its habitat, we evaluated botanical features, ontogenetic phases, the ecological and phytocenotic structure of the rare and endangered of Rhodiola rosea L. (golden rose root) populations from the highlands of Eastern Kazakhstan. R. rosea in the study region lives on damp mossy rocks, rocky slopes, overgrown moraines and along the banks of mountain rivers in the upper limit of cedar-larch forests, subalpine and alpine belts, in the altitude limit of 1700–2400 m. In the studied region, R. rosea begins to vegetate in May–June, blooms in June–July, the fruits ripen in August. The species is encountered in the high mountain ranges of the Kazakh Altai and Saur-Tarabagatai. Unfavorable habitat conditions for the species are overgrown by sedge-grass and birch-moss communities. The most common species at sites with R. rosea are: Schulzia crinita, Achillea ledebourii, Doronicum altaicum, Macropodium nivale, Hylotelephium telephium, Rhodiola algida, Carex capillaris, C. aterrima. Ontogenetic study revealed that all age-related phases were present, with the exception of the senile states. Individual life expectancy shown to be 50–55 years. The analysis of the species composition in the communities with R. rosea showed that the leading families in terms of the number of accompanying species are Poaceae, Ranunculaceae, Asteraceae, Rosaceae and Caryophyllaceae, Apiaceae, Fabaceae; while the most dominant genera are: Carex, Aconitum, Dracocephalum, Festuca, Pedicularis, Poa, Salix; the ecological groups are dominated by psychrophytes, mesophytes mesopsychrophytes; the Asian, Eurasian, and Holarctic groups are the most represented groups. Dominant life forms according to Serebyakov were rod-rooted, brush-rooted, short-rooted and long-rooted grasses, while based on Raunkiaer’s groups the overwhelming majority consisted of Hemincryptophytes (74%). The R. rosea populations of Kazakhstan represent an important gene stock of the species. Our study provides new insights into the species’ biology thus contributes to the conservation of biodiversity on a wide spatial scale.


Background
The study of the ecological and botanical characteristics of natural populations of rare and vulnerable plants remains a priority in the strategy of biodiversity conservation, especially if they are Wild Relatives of crops (Perrino and Wagensommer 2021). Currently, many valuable medicinal plants are subjected to spontaneous gathering, Open Access *Correspondence: mzhakypzhan@mail.ru 2 Al-Farabi Kazakh National University, Almaty, Kazakhstan Full list of author information is available at the end of the article This article belongs to the Topical Collection: Ecology. as a result of which the number and areas of natural habitats are decreasing, the natural balance in communities is disrupted, which leads to population degradation (Cunningham et al. 2020).
This concerns R. rosea, the demand for which has grown significantly throughout the world in recent years, which threatens the extinction of natural populations on a global scale. The global demand for R. rosea raw material will continue to grow (Bernard 2016), which could lead to catastrophic consequences. The main drivers of the increased demand for R. rosea raw materials are the expansion of the range of drugs, dietary supplements, and cosmetics containing R. rosea (Brinckmann et al. 2020). A highly important priority is to preserve the natural environment in which R. rosea grows and it is a high priority is to evaluating the type and flock size of the grazing in order to preserve their natural habitat, using sustainable criteria Buse et al. 2015). Nowadays R. rosea is classified as rare and endangered species, in many regions-as a protected plant. The growing demand increases the load on the natural populations of the species. As a result, it has become a threat object in many Eurasian countries, including the Czech Republic (Grulich 2012), Slovakia (Ferakova et al. 2001), Bosnia and Herzegovina (Platikanov and Evstatieva 2008), Bulgaria (Tasheva and Kosturkova 2012), Germany (Metzing et al. 2018), Austria (Niklfeld and Schratt-Ehrendorfer 1999), the Russian Federation (Trutnev et al. 2008), Mongolia (Urgamal 2018), China (Qin 2017).
One reliable way to preserve this species is to introduce it into cultivated culture (Karpukhin and Abramchuk 2020). The scientists suggest that in situ protection is insufficient to preserve the gene pool of the R. rosea population. It seems appropriate to create ex situ populations and return them to nature (Hou and Lou 2011). It is successfully cultivated in botanical gardens and in research institutes of plant growing in Russia (St. Petersburg, Gorno-Altaisk, Novosibirsk, Irkutsk, etc.) (Moryakina et al. 2008) as well as in European countries: Bulgaria (Platikanov and Evstatieva 2008), the United Kingdom (Peschel et al. 2018), Poland (Buchwald et al. 2015), Italy (Fusani 2019).The regenerative capacity of wild plants is limited due to the very low seed germination rate (5-35%) and the coefficient of vegetative reproduction (Platikanov and Evstatieva 2008). In view of the above, the widespread use of natural specimens in many countries has led to the disappearance of the species, which has provoked the adoption of a number of conservation measures: cultivation in appropriate conditions (Matthys et al. 2007). In many countries, sustainable ecological use of natural resources, the conservation and the preservation of natural areas as a special environmental activity are regulated (Yaneva et al. 2020).
Despite the great interest in the R. rosea and extensive research in the field of phytochemistry, plant bio-technology remained less researched and widely used (Li et al. 2019;Olsson et al. 2009). The stages of morphogenesis of the species and the maximum age of wild populations of R. rosea are also poorly studied (Brinckmann et al. 2021). The studies of many Russian scientists are devoted to the study of ecological and botanical proper-ties, distribution, ontogeny of populations (Sofronov et al. 2016;Valuiskih et al. 2017;Panossian et al. 2010;Yakubov et al. 2019;Shadrin et al. 2020). A relatively fewer population ecological studies of R. rosea were performed in Europe and North America (Olfelt et al. 2014;Aiello et al. 2013), however, the research on genetic diversity and phylogeography of populations are well represented (György et al. 2012;György et al. 2013;György et al. 2014;Soni et al. 2010;Kozyrenko et al. 2018).
Rhodiola rosea L. (Crassulaceae DC) has a wide circumpolar distribution in the northern hemisphere from the low-Arctic to high-temperature regions of Asia, Europe, and North America (Cuerrier 2014;Brinckmann et al. 2021). It is a rare species included in the Red Book of Kazakhstan (2014) with III class status and is regarded as a threatened species. According to the data of International Union for the Conservation of Nature Resources (IUCN), the rarity category is Least concern (LC) (Chadburn 2020). It is guarded in Katon-Karagai State National Natural Park, Markakol and Eastern Altai protected area in the researched region. It grows in alpine and subalpine vegetation belts, stony tundra's, on the rocks and rocky hills, on placers and moist soils along river banks. In Kazakhstan, it has been documented in three floristic regions: Altai, Tarbagatai, Dzhungarskiy Alatau (Vasilyeva 1961). Distribution area is in the Southern Siberian mountains, in the Ural, in transpolar regions of Yakutia, in the highland areas of Eastern Siberia and Far East, on the coasts of White and Barancevo seas, in Mongolia, China, North America and in Asia Minor (Borisova 1939;Vasilyeva 1961;Ivanova 1979;Peshkova 1994).
Therefore, purpose of the work is to study distribution and density of R. rosea populations in East Kazakhstan, floristic and habitat characteristics, including variation in ontogenetic states of the individuals.

Materials and methods
The Kazakhstan part of Altai is a system of ridges in the southern and the southwestern part of Altai, as a mountainous country that stretches from south to north and from west to east for almost 400 km. It is a part of the southwestern periphery of the Altai-Sayan mountain system and with its inherent structure of landscape and high-altitude zones. According to the physical and geographical conditions, the territory of the Kazakhstan Altai is subdivided into three subdistricts which are Southwestern Altai, Southern Altai, Kalbinsk Highlands (Yegorina et al. 2003).
The research was carried out between 2015 and 2020 in Southern Altai (Narym, Sarymsakty, Southern Altai Tarbagatai, Kurchym ridges) and Western Altai (Ivanov, Ubi, Ulbi, Koksim Linei, Western Listvyaga ridges) of Kazakhstan part. The investigated region administratively belongs to the East Kazakhstan region. The geographic zoning and names of mountain ridges are specified according to the Physical Map of Kazakhstan. To identify the phytocenotic features of R. rosea populations, the traditional methods of field geobotanical studies were perfumed using the ecological-physiognomic approach. The ecological and physiognomic types combine plant communities with dominants belonging to one ecobiomorph and ecologically similar groups of species (Bykov 1970).
The description of the populations was carried out using special description forms. In each population, 15 study sites were laid, the area of the site was: 10 × 10 m (100 m 2 ). A total of 150 sites were taken into account. The GPS device recorded the marginal points of the community boundaries to identify the area. First, general information was written in the form: description number, geographical location, date, coordinates, height, site size, photo number, then the following main sections are reflected in the form: The name of the vegetation type based on dominant species; the floral composition of the community with an indication of occurrence of species. To do this, we selected a section in the redevelopment of a homogeneous contour. The GPS device determined the coordinates and the absolute height.
The rarity category and status of the species are indicated in accordance with the Kazakhstan Red Data Book (Kazakhstan Red Data Book 2014) and The IUCN Red List of Species (IUCN 2020). Uranov (1969) method was applied for the purposes of researching the life cycles. The methodological guidelines developed by Golubev and Molchanov (1978) were used as a basis for studying ecological, biological properties of the species in the reallife field conditions.
The spectra analysis of geographic elements of floras of various ranks, including floras of plant communities in the scope of specified classification units (composition of the floras) is one of the main tools of comparative floristical studies. Composition of the flora is a set of plant species which form communities of any rank and any type of vegetation. From this point of view, the composition of the flora represents the unification of historically and coenotically homogeneous groups of species within the syntaxon, which makes them the most important indicator of the vegetation cover from the level of a particular phytocenosis to altitudinal-belt units. The life forms of plants of the floristic composition of R. rosea communities are given according to the classifications of Serebryakov (1962) and Raunkiaer (1934). Species composition in R. rosea communities by ecological groups and area of species are given according to the classification of Kuminova (1960).
Plant names are listed according according to Plants of the World Online (POWO 2021). The analysis of species' floral composition in the surroundings of R. rosea was carried out in comparison with the Alpine flora of Altai (AFA) (Revushkin 1988).
According to the number of fibers remaining annually after the death of the shoots on the R. rosea rhizome, it is possible to judge the age of plants. In any cenosis, as a rule, all species are represented by numerous individuals of various ages, from seedlings to old plants. We studied all individuals of the species in different populations and established the course of accumulation of fibers with age, and based on this we calculated the age of individuals. Undoubtedly, the determination of the age of individuals by this method may be inaccurate, since some of them are characterized by a very slow course of development, while others, on the contrary, develop very quickly. However, the accuracy of age determination increases with an increase in the number of studied individuals. We determined the age of 100 individuals from different populations.
Based on the long-term herbarium collections of the authors of this work stored in Altai Botanical Garden (further referred to as ' Alt. ') and Astana Botanical Garden (NUR), as well as a result of the revision of the herbarium materials of the Moscow State University (MW) (Seregin 2020) and the herbarium of the Institute of Botany and Phytointroduction (AA), the distribution of R. rosea in Eastern Kazakhstan was revealed. In addition, the following literature were taken into account: Artemov 2020;Kotuhov 2005;Isayev 1993;Zibzeyev and Nedovesova 2015;Kubentayev et al. 2018, Kubentayev et al. 2019. The distribution map is shown in Fig. 1.
The correlation analysis was calculated using the Rstudio software (Rstudio Team 2015). Statistics of morphometric parameters were performed using the program Statistica 10 (StatSoft STATISTICA 10 2011).

Distribution of R. rosea in East Kazakhstan
R. rosea is distributed on the ridges of the Kazakhstan Altai and Tarbagatai in the East Kazakhstan region. The species is found in the following administrative districts: Katon-Karagaysky, Kurchumsky, Riddersky, Glubokovsky and Urdzharsky districts. The examined herbarium samples are given below.

Population (Macropodium nivale -A ngelica arch-
angelica -R. rosea ) is timed to the western slopes of shallow ravines of shallowed river beds (Fig. 3  among the shrubs, which forms separate small patches. Favorable water and temperature conditions, high humus content in the soil determine the lush development of vegetation, which negatively affects the populations of R. rosea due to its low competitiveness. 3. Population (Alchemilla gottsteiniana -Polygonum ellipticum + R. rosea ) is timed to smooth slopes of alpine meadows on the meadow soils (Fig. 3) Altai. According to the data obtained, the highest number of individuals per 1 m 2 was observed in P10 (0.75), P7 (0.68) and P1 (0.56), a relatively low number per unit area was noted in P8 (0.18 ), P 6 (0.21) and in P3 (0.23). R. rosea populations with a high number of individuals per unit area, are composed of mostly undersized, multi-shoot shrubs having large flowers. In the population with a low abundance of Rhodiola specimens per unit area, tall individuals are observed, with loose, low-shoot bushes and relatively small flowers. R. rosea populations growing in habitats with a high number of accompanying species are multi-stemmed and have large flowers. Populations with a low number of individuals, are characterized by tall, crumbly, low-running bushes and relatively small inflorescences. This pattern is explained by the habitat conditions. As a rule in the forest belt and in tall grass communities the specimens of R. rosea are relatively tall (45-50 cm), have crumbly low-running bushes (6-10 pcs) and small inflorescences (3-4.5 cm). In open, poorly populated areas, and along the valleys of mountain streams, individuals are significantly undersized (20-25 cm), but have a multistem structure (30-50 pcs) and develop large flowers (5.2-6 cm) (Fig. 4).

Phenology of R. rosea in East Kazakhstan
R. rosea begins to vegetate under snow cover starting from mid-May to mid-June in the studied area and when the snow melts, it begins to grow rapidly. Flowering period lasts from mid-June to late July. The fruits ripen from August to September. It should be noted that the seasonal rhythm of the species' development depends on the height of the location of the population. The species begins to grow from mid-May in the upper limit of the green belt at an altitude of 1700-1900 m. R. rosea grows in the second half of June in the alpine belt at an altitude of 2200-2400 m. On average, the growing season lasts 4 months.
The species in the surveyed area reproduces predominantly in a vegetative way due to the division of rhizomes that are spreading by the melt water during the period of abundant snow melting, but in some locations seed renewal is noted. Seeds are small, elongated. The seed shape is curved-pin-shaped. The seeds surface is bare, longitudinally wrinkled. The seed scar is small, slightly protruding, basal, rounded. Seed color is from light brown to hazel. The length is 2.13 ± 0.16 mm, Cv = 15.7%; min-max -1.65 -2.78 mm, the width is 0.48-0.81 mm (0.59 ± 0.07 mm, Cv = 17.5%). The weight of 1000 seeds is 0.208-0.239 g. Once in the soil, ripened seeds undergo natural stratification over the next 7-8 months. Germination rate of R. rosea seeds in lab conditions at 18° C in three replications was 51%. Plantlets ( Fig. 5(p)) appear at the end of May and beginning of June. Emergence of seeding is epigeous. The cotyledons are light green, bare, succulent 3,2±0.09 mm long, 1.8 ± 0.06 mm wide. The leaf blade is ovalovoid, have short petiole up to 2.8 mm long, rounded at the apex, sharply tapered at the base turning into a short petiole. The hypocotyl is 3.2 ± 0.07 mm long, up to 1.1 ± 0.03 mm thick, pale green, the basal part is thickened, sharply passes into the embryonic (primary) root. The main root reaches 2.3 ± 0.08 cm by the time the cotyledons dry up. Cotyledons persist until mid or late July. In the second and the third months after germination of the seed, this age condition ends.
At the end of July and beginning of August the species pass into juvenile state (Fig. 5j). Plants in this phase are characterized by the formation of leaf rosette of 2.6 ± 0.04 leaves, the presence of a crown bud and 1-2 auxillary buds of an open type. The part of the growth sprout (rhizome) does not die off after the end of the vegetation season, but becomes perennial, from which the rhizome is subsequently formed. The seedlings end the juvenile phase at the age of 2-3 years old and less often.
Immature phase (Fig. 5(im)) is characterized by the growth of vegetative stems of normal type in the structure of medial sprout. The medial sprout of 7,2±0.12 cm height has 6,9 ± 0.21 pieces of natural leaves. The leaves are set by turn, the leaf blade is elongated-ellipsoid at the base and smoothly tapers into a short petiole. The crown and lateral buds of the growing sprout are of a closed type. The growth part of the rhizome is 2.4 ± 0.2 cm in length and 0.6 ± 0.03 cm in thickness. Rhizome branching is observed. The root system is well developed, in the horizontal projection is 2.8 ± 0.08 cm and in the vertical projection 12.1 ±  1.56 cm (deepened). In the primordial state, plants sustain on average 2 vegetation seasons. In the future, the plants move to the next age state. Virginal phase (Fig. 5(v)) might be seen for 5-7 year and starts with the branching of the medial sprout of branching of the medial sprout of the rhizome with the development of a significant number of lateral sprouts of the first order with the development of stems on them. The plants in this age state are 14 ± 1.31 cm in height. The rhizome has 3,6 ± 0.07 pieces of stems of the first order. The medial sprout of the rhizome and some lateral sprouts are 6.2 ± 0.06 cm long at the base and 0.83 ± 1.2 cm across. The root system is well developed, 10.2 ± 1.8 cm in horizontal projection and 16 ± 2.2 cm in vertical projection. The primary root is about 1.8 ± 0.06 cm thick.
The plants start the young generative phase (Fig. 5(g1)) at the age of 8-11 years. Generative stems are formed in the sprout. Young plants usually generate 2.8 ± 0.06 pieces of generative stems with a depleted inflorescence of 1.8 ± 0.02 flowers and 8,2 ± 2.6 vegetative stems in their first two years. Plants at the age of 8-10. more often at 12 years begin to form generative stems on the sprouts of the rhizome of the first type. The rhizome of the horizontal projection is 3.2 ± 0.9 cm thick and 16.3 ± 0.8 cm long. Plants are dioecious. The flowers are unisexual, yellow, four-membered (less often five-membered), small (3.2 ± 0.06 mm long), collected in the final dense multi-flowered corymbose inflorescences. The number of flowers in an inflorescence are 6.3 ± 1.8 having a diameter of 4.8 ± 0.35 cm. The height of the plant is 30 ± 1.8 cm. In this age state, stems begin to form on the sprouts of second-order rhizomes. This age-related condition ends at 18-22 years.
The mature generative individuals (Fig. 5(g2)) include plants of 22-30 years, characterized by a powerful development of 43 ± 2.1 cm in height. In this state there is an intensive development of the stems on the medial sprout of the rhizome and sprouts of the first and second types. Such individuals have 35 ± 3.6 pcs of generative and 42 ± 2.8 pcs of vegetative stems. The inflorescence has 10.6 ± 1.7 flowers being 5.2 ± 0.35 cm in diameter. In this phase abundant flowering and fruiting and clone formation was observed. Particulation and clone formation are observed.
The plants start old generative phase (Fig. 5(g3)) at the age of 30-40 years. In this state, there is a noticeable predominance of the vegetative stems up to 68 ± 3.9 pcs and the formation of a significant number of weakened generative stems 52 ± 2.8 pcs. The inflorescence is composed usually of 6.2 ± 0.12 flowers, having 3.8 ± 0.35 cm in diameter. We observed here the beginning of the rhizome sprout decline and the formation of extensive foci of the main root necrosis and medial sprout of the rhizome. Also, mass dying of rhizomes' sprout of the first order is typical in this age phase.
Ageing individuals (Fig. 5(a.i.)) are very rare. It is pretty hard to define the age of this species. According to our data, specimens start this phase at the age of 50-55 years. In this state, extensive foci of necrosis appear almost along the entire length of the main rhizome and its disintegration into separate girders can be seen. There are frequent cases of dying of the adventitious roots and of the first-order rhizome sprouts. The bushes easily break

Floristic composition of the R. rosea communities
As a result of data processing of our field studies and herbarium collections, in the floristic composition of R. rosea communities we registered 140 species belonging to 39 families and 104 genera, which is 14% of the Altai highland flora (AHF), where 996 species of vascular plants from 325 genera and 80 families are registered ( Table 2) (Fig. 6). They account for 77 (55%) species of the flora composition.

Discussion
In the studies of Vedernikova and Nikandrova (2000) interesting differences was found between two groups of populations R. rosea, when studying interpopulation variation which are populations timed to the mountain tundra belt on the one hand, and populations timed to near-snowy lawns and river meadows on the other hand. In the second group of populations, a tendency towards a clear predominance of features which characterize the general habit of the plant (height of sprouts, their number, leaf size, etc.) is noted. Therefore, these environmental conditions can be considered optimal for R. rosea. Our studies also confirmed this theory. In open, sparsely populated areas and along the valleys of mountain streams, individuals are significantly stunted (20-25 cm), but have multi-stemmed bushes (30-50 pcs) and large inflorescences (5.2-6 cm in diameter). The height of R. rosea individuals in the examined populations ranges from 24.42 to 49.29 cm, the diameter of the inflorescence varies between 3.21 and 5.25 cm. These parameters are within the normal range for the species (Eggli 2003).
In the studied region, R. rosea is found in different ecological conditions and altitudinal ranges of 1700-2400 m (from the upper limit of the forest belt to the tundra belt of the mountains). The wide range of habitats of the species is also confirmed by other researchers (Allen et al. 2014;Cuerrier and Ampong-Nyarko 2014;Fu et al. 2001).
Based on our study we can confirmed that R. rosea is present in the Southwestern and Southern Altai of the studied region, as well as on the Tarbagatai ridge. When reviewing the herbarium collections of AA, one herbarium collection was found from the territory of the Kalbinsky ridge (Koktau mountains under the ridge of rocks 1440 m, singly, Snegirev V., 31. V. 1994 (AA)), but our studies did not confirm this location of the species. Perhaps an error could have been made in labeling, or this collection may be evidence of the former distribution of the species preserved in the relict complex "Sinegorskaya fir Grove" (Myrzagalieva 2006).
R. rosea plants are known to be long-lived perennials, but maximum ages in wild populations have not been studied (Brinckmanna et al. 2021). According to Nekratova and Nekratov (2005), the maximum age of R. rosea is 80 years. Our researches allowed us to establish the maximum age of R. rosea in the studied region at 55 years, after which the bushes easily disintegrate into 3-6 clones, forming new plants and spreading over the population area.
According to Revushkin (1988), 996 species of vascular plants from 325 genera and 80 families were registered in  the AHF, the species diversity of the floral composition of R. rosea communities was 140 species, which is 14% of the AHF flora. The composition based on the 10 most frequent families in terms of their quantities is almost identical to the composition of the leading families in AHF. However, their arrangement in descending order is somewhat different, which is due to the allocation of R. rosea to the redivided ecological groups. Communities with R. rosea are dominated by single and two species families, which characterizes the AHF flora (Sofronov et al. 2016), the Western Sayan (Kamelin 2016) and the arctic floras which develop in extreme habitat conditions and indicates the complicacy of the floral genesis process (Sofronov et al. 2016). The analysis of the genera spectrum accords well with the composition of the AHF leading genera (Revushkin 1988), where Carex, Aconitum, Dracocephalum, Festuca, Pedicularis, Poa, Salix genera prevail.
According to Revushkin (1988) it is enough to consider in the ecological analysis of highlands the humidity and the type of the substrate. No need of taking into account the temperature, the salinity or the soil fertility. However, we consider that the classification of ecological groups presented by Kuminova (1960) is well applicable for the highland flora analysis and the temperature regime is the crucial feature of the highlands. Despite that, we agree that it is inappropriately to allocate groups due to their salinity and soil fertility. The  data showed that the composition of the flora is dominated by psychrophytes, mesophytes, mesopsychrophytes and mesoxerophytes. These groups account for 109 (78%) species of the total floristic composition of the R. rosea communities. In general, this distribution of species by ecological groups is considered characteristic for the flora of the highlands. The percentage of the chorological groups differs somewhat from the AHF, however, the leading groups nevertheless converge. It should be noted that a significant excess of the Eurasian group was detected, (30%) compared to the AHF (14.4%), and the participation of Asian species is in the opposite way lower (39%) than the AHF (62%). The distribution of species by life forms is in good agreement with the AHF flora.

Conclusions
In general, the state of R. rosea populations in the studied region should be considered satisfactory. In the surveyed populations anthropogenic influence was not observed, apparently because of difficult accessibility of the habitats. The main limiting factor is the low competitiveness of the species within the communities. When the habitats are overgrown with herbaceous-grass vegetation, populations of R. rosea are forced to gradually migrate to less overgrown areas, or fall out of the phytocenosis. All surveyed populations are maintained mainly by vegetative reproduction. Seed renewal is low as a result of seedlings dying in the early stages of development due to extreme habitat conditions.